B

Fig. 4. (A) Copper-transport hepatocytes. The hepatocyte is the main regulator of copper efflux from the body, by means of copper transport across the biliary canalicular membrane (apical surface) of the hepatocyte. ATP7B is the main regulator of this process. When the liver copper concentration is low, ATP7B is located in the TGN, where it supplies copper to ceruloplasmin, which is secreted into the blood. If copper levels start to rise in the cytoplasm because of excess copper intake in the diet, ATP7B is induced to traffic to a vesicular compartment and possibly also to the apical membrane, pumping copper into vesicles that later discharge the copper into the bile. APT7B on the apical membrane may also pump copper directly out of the cell. If the dietary intake of copper is so high that the ATP7B-mediated efflux is insufficient, copper accumulates and induces metallothioneins. Some of this Cu-MT is taken up by lysosomes and some may be discharged by exocytosis into the bile. (B) Copper transport in hepatocytes of an individual with Wilson's disease. ATP7B is inactive, so no copper is incorporated in ceruloplasmin, leading to secretion of apoceruloplasmin (continued)

Fig. 4. (A) Copper-transport hepatocytes. The hepatocyte is the main regulator of copper efflux from the body, by means of copper transport across the biliary canalicular membrane (apical surface) of the hepatocyte. ATP7B is the main regulator of this process. When the liver copper concentration is low, ATP7B is located in the TGN, where it supplies copper to ceruloplasmin, which is secreted into the blood. If copper levels start to rise in the cytoplasm because of excess copper intake in the diet, ATP7B is induced to traffic to a vesicular compartment and possibly also to the apical membrane, pumping copper into vesicles that later discharge the copper into the bile. APT7B on the apical membrane may also pump copper directly out of the cell. If the dietary intake of copper is so high that the ATP7B-mediated efflux is insufficient, copper accumulates and induces metallothioneins. Some of this Cu-MT is taken up by lysosomes and some may be discharged by exocytosis into the bile. (B) Copper transport in hepatocytes of an individual with Wilson's disease. ATP7B is inactive, so no copper is incorporated in ceruloplasmin, leading to secretion of apoceruloplasmin (continued)

when copper levels are high in an enterocyte, ATP7A will be located on the basolateral membrane, presumably increasing copper uptake into the circulation. As this is contrary to the observed decrease in copper uptake when dietary levels are high, it may suggest that primary control over the amount of dietary copper absorbed is the modification of uptake across the apical surface, as discussed earlier. Nevertheless, the Cu-induced trafficking behavior of ATP7A in intestinal cells has not been reported and may show some differences from that observed in nonpolarized cells, so a possible role for ATP7A in regulating uptake cannot be excluded.

The liver is the primary regulator of copper status and this is achieved by modulation of the rate of biliary excretion, mainly mediated by ATP7B (Fig. 4A). This protein also delivers copper to cerulo-plasmin in the TGN, thus explaining why patients with Wilson's disease have low biliary excretion of copper and usually have low levels of holoceruloplasmin in plasma (Fig. 4B). The liver removes excess copper by excretion in the bile and this mechanism is an important regulatory step in cases, such as an acute exposures to high copper, where the small-intestine regulatory mechanisms are insufficient or are too slow (e.g., induction of MT) to exclude the excess copper. The increased knowledge of the cell biology of ATP7B, discussed in Section 6., suggests that the increased copper efflux when the cell is exposed to excess copper is due to copper-induced trafficking of ATP7B to vesicles and/or the apical membrane of the hepatocyte, resulting in the efflux of copper into the bile. This efflux can be achieved either by exocytosis of the copper-laden vesicles or direct pumping of copper across the biliary canalicular membrane (39), (Fig. 4A). Lysosomal exocytosis into the biliary canaliculae is thought to form part of the pathway for biliary excretion of copper (40), but it is not clear whether these lysosomes described in this study correspond to the copper-loaded vesicles noted earlier. The exact details of this process are unclear, as there is still debate on whether ATP7B actually reaches the apical membrane (41,42); nevertheless, the trafficking response of the protein is clearly the fundamental copper-sensing mechanism ultimately responsible for the increase in biliary copper excretion in copper-loaded animals.

When the dietary intake exceeds the capacity of the biliary excretion mechanisms, for instance as a result of dietary supplementation (43) or in Wilson's disease (44) (e.g., Fig. 4B), much of the excess metal is associated with MTs. In such copper-loaded livers, the copper also accumulates in lysos-omes (43). Glutathione appears to be involved in the biliary excretion of copper in copper-loaded livers (39).

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